Immunotherapy represents an important treatment option for patients with metastatic melanoma but response rates remain low. Multiple infusion high dose IL-2 therapy to directly boost immunity represents one such option, but it is complicated by high and severe toxicity and by concurrent expansion of Tregs. Newer options are anti-CTLA4 (ipilimumab) and anti-PD-1 (nivolumab) therapy that blocks negative immune regulation to induce an anti-tumor response. These therapies shows promise by reviving pre-existing CTLs to respond to the tumor and have helped to renew interest and enthusiasm for immunotherapy of cancer in general. This approach is sometimes accompanied by sides effects such a colitis, autoimmune hypophysitis, and severe cutaneous skin reactions. More importantly, many patients still do not respond to this therapy. An ideal therapy for some cancers, including melanoma, may be to capture the beneficial features of IL-2 to boost immunity and immune memory of CTL and those of negative checkpoint inhibitors, such as anti-CTLA4 or anti-PD1, to further drive anti-tumor immune responses. However, for this to become a reality, the use of IL-2 must be repurposed to reduce toxicity and minimize effects on Tregs. New findings from my laboratory may achieve this goal. In brief we have learned that transient application of IL-2, in the form of agonist IL-2/anti-IL-2 complexes or with a novel IL-2 fusion protein (IL-2FP) developed in my lab only transiently affects Tregs while highly boosting T effector (Teff) and long-lasting memory responses. This approach required much lower levels of IL- 2 that should minimize non-specific toxicity associated with high dose IL-2. Administering anti-CTLA4 or anti- PD1 during IL-2 therapy, particularly in the context of a tumor vaccine, may direct robust responses toward the tumor, leading to increased response rates, and minimize off target self-reactive T cells. The main hypothesis of this proposal is that endogenous or vaccine-induced anti-tumor immune responses will be greatly enhanced by transient administration of IL-2FP alone or in conjunction with the negative checkpoint inhibitors, anti-CTLA4 or anti-PD1. This hypothesis will be tested in mice using the weakly immunogenic B16 melanoma model because several tumor antigens have been defined, which facilitate production of relevant tumor vaccines and identification of endogenous tumor-reactive T cells, and because CD8+ TCR transgenic T cells, specific for B16 tumor antigen gp100, are available to facilitate mechanistic studies. To test this hypothesis we propose the follow aims. 1) To assess the extent that IL-2FP boosts tumor immunity toward the B16 melanoma in C57BL/6 mice directly or in the context of tumor vaccines. The effect of the negative checkpoint inhibitors, anti-CTLA4 or anti-PD1, will be tested on IL-2-dependent tumor responses. 2) To evaluate the mechanisms by which IL-2FP alone or in combination therapies leads to tumor immunity. This includes evaluating the extent CD8+ T cell intra-tumor immune responses are enhanced.